Vibrio cholerae is the causative agent of the severe diarrheal disease cholera, which affects millions of people worldwide each year. The goal of our research is to understand the mechanisms for controlling expression of the genes involved in V. cholerae virulence. The V. cholerae virulence genes include those encoding cholera toxin (CT), which produces the characteristic symptoms of cholera, the toxin co-regulated pilus (TCP), which is involved in the colonization of the small intestine of the host, together with a collection of other genes whose roles in virulence are poorly understood. Expression of the virulence genes is controlled by a network of transcriptional regulators; however, one protein, ToxT, ultimately activates transcription of nearly all the virulence genes, and understanding how this protein functions is the focus of our work. Experiments described in this application will examine the following: (1) the amino acid residues in ToxT that are involved in transcriptional activation and DNAbinding will be determined using a genetic selection/screening followed by further characterization of mutants in vivo and in vitro. (2) There is mounting evidence that ToxT activity is regulated post-transcriptionally. We will determine how the post-transcriptional control of ToxT is manifested, examine environmental ToxT variants for functionality in epidemic V. cholerae, and assess the effects of constitutive ToxT expression in vitro and in an animal model. (3) We will determine the identities of all proteins whose expression is ToxT dependent by comparing proteomic analysis of V. cholerae having wildtype toxT and V. cholerae having a toxT deletion. These experiments should significantly increase our knowledge of ToxT protein function and of the functions of genes whose expression is activated by ToxT, and could lead to new therapeutic targets for cholera disease. Cholera remains a significant worldwide problem, particularly in areas suffering from the effect of war, natural disaster, or economic hardship, and improved treatments should greatly increase the quality of life for people in these regions. [unreadable] [unreadable] [unreadable]